U.S. patent number 8,576,395 [Application Number 13/739,730] was granted by the patent office on 2013-11-05 for integrated microbial collector.
This patent grant is currently assigned to Azbil Biovigilant, Inc.. The grantee listed for this patent is BioVigilant Systems, Inc.. Invention is credited to John Y. Babico, Jian-Ping Jiang.
United States Patent |
8,576,395 |
Babico , et al. |
November 5, 2013 |
Integrated microbial collector
Abstract
A system for real-time sizing of fluid-borne particles is
disclosed. The system further determines, in real time, whether the
detected particles are biological or non-biological. As the fluid
is being tested, it is exposed to a microbe collection filter which
is cultured to determine the type of microbes present in the fluid
being tested.
Inventors: |
Babico; John Y. (Tucson,
AZ), Jiang; Jian-Ping (Tucson, AZ) |
Applicant: |
Name |
City |
State |
Country |
Type |
BioVigilant Systems, Inc. |
Tucson |
AZ |
US |
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Assignee: |
Azbil Biovigilant, Inc.
(Tucson, AZ)
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Family
ID: |
42266673 |
Appl.
No.: |
13/739,730 |
Filed: |
January 11, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20130130368 A1 |
May 23, 2013 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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12642722 |
Dec 18, 2009 |
8358411 |
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61138878 |
Dec 18, 2008 |
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Current U.S.
Class: |
356/335; 356/338;
435/288.7; 356/336; 435/34 |
Current CPC
Class: |
G01N
15/1459 (20130101); G01N 21/6486 (20130101); G01N
21/49 (20130101); C12Q 1/04 (20130101); G01N
15/14 (20130101); G01N 21/64 (20130101); G01N
2015/1493 (20130101); G01N 2015/0088 (20130101); G01N
2015/0065 (20130101) |
Current International
Class: |
G01N
21/00 (20060101); G01N 15/02 (20060101); C12Q
1/04 (20060101); C12M 1/34 (20060101) |
Field of
Search: |
;356/335-343,72-73,317,218 ;250/461.1,461.2,458.1,286,287 ;436/164
;435/288.7 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: Nguyen; Sang
Attorney, Agent or Firm: Michael Curley Quarles & Brady
LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This is a Continuation Application which claims priority from U.S.
Non-Provisional Application having Ser. No. 12/642,722, filed on
Dec. 18, 2009, to issue as U.S. Pat. No. 8,358,411, which in turn
claims priority to U.S. Provisional Application having Ser. No.
61/138,878, filed Dec. 18, 2008. The contents of each of these
applications are entirely incorporated herein by reference in their
entirety, for all purposes.
Claims
What is claimed is:
1. A microbial detection and identification system, comprising: a
housing, including a sampling area having a fluid, a light source,
a first optical detector and a second optical detector; a blower in
fluid communication with said housing, wherein said blower supplies
negative pressure to said housing thereby drawing environmental air
into said sampling area and evacuating air from said sampling area;
a microbe collection filter in fluid communication with said
sampling area, wherein environmental air is drawn by the blower
from said sampling area through the microbe collection filter, and
a perforated support plate in contact with a rear side of the
microbe collection filter.
2. The system of claim 1, wherein said sampling area is defined by
an input nozzle and an output nozzle.
3. The system of claim 2, wherein said microbe collection filter
includes a front side facing said exit nozzle, a rear side in fluid
communication with said blower, and a gas permeable interior
allowing said fluid to flow into said front side and out of said
rear side.
4. The system of claim 1, wherein, said light source illuminates
particles in said sampling area, said first detector detects light
scattered into a predetermined angular range by particles of a
predetermined size, said second detector measures light emitted by
fluorescence from illuminated biological particles in said sample
area, and said fluid is exposed to said microbe collection filter
causing particles in said fluid to adhere to said microbe
collection filter.
5. The system of claim 4, further comprising scattered light
collection components that direct scattered light from said
sampling area to said first detector and a plurality of optical
components that direct fluorescence light from said sampling area
to said second detector.
6. The system of claim 5, wherein said plurality of optical
components includes a long-pass filter that selectively transmits
light having a wavelength of light emitted by fluorescence by
illuminated particles in said sample area.
7. The system of claim 6, wherein said long-pass optical filter
comprises two long-pass reflective filters in series.
8. The system of claim 5 further comprising an ellipsoidal
reflector having a first focus at said sampling area and a second
focus near said second detector.
9. The system of claim 1, wherein said light source comprises an
LED or a diode laser, and wherein said light source emits at a
wavelength of approximately between 350 nm and 410 nm.
10. The system of claim 1, wherein said blower is in fluid
communication with said microbe collection filter, and wherein air
is drawn through said microbe collection filter after it is
optically measured.
11. The system of claim 1 wherein said microbe collection filter
comprises a gelatin wafer.
12. The system of claim 1, wherein said microbe collection filter
has a front side arranged transverse to the flow of fluid being
measured.
13. A microbe collection filter for use with a microbial detection
and identification system, the system having a housing, including a
sampling area having a fluid, a light source, at least one optical
detector, and a blower in fluid communication with said housing,
wherein said blower supplies negative pressure to said housing
thereby drawing environmental air into said sampling area
evacuating air from said sampling area; the microbe collection
filter having a front side, a rear side, and a fluid-permeable
interior, wherein the microbe collection filter is arranged
transverse to the flow of fluid being measured, wherein the microbe
collection filter is supported by a perforated support plate in
contact with the rear side of the microbe collection filter, and
wherein the perforated support plate is arranged between the
microbe collection filter and the blower.
14. A microbial detection and identification system comprising: a
housing, including a sampling area having a fluid, a light source,
at least one optical detector, and a blower in fluid communication
with said housing, wherein said blower supplies negative pressure
to said housing thereby drawing environmental air into said
sampling area evacuating air from said sampling area; a removable
cartridge housing a microbe collection filter having a front side,
a rear side, and a fluid permeable interior volume, and an input
aperture arranged transverse to the flow of fluid being measured in
fluid communication with both the sampling area and the front side
of the microbe collection filter, wherein the cartridge includes a
perforated support plate in contact with the rear side of the
microbe collection filter, and an output aperture arranged between
the rear side of the microbe collection filter and the blower.
Description
FIELD OF INVENTION
The present invention relates to generally to a system and method
for detecting airborne or liquid-borne particles (generally,
fluid-borne particles), and more particularly to a system and
method for detecting airborne or liquid-borne particles,
determining the size of the particles, determining the particles'
status as biological or inert, and classifying the type of biologic
particle detected.
BACKGROUND OF INVENTION
A variety of manufacturing environments require strict control over
the presence of foreign debris in the air. Semiconductor
manufacturing, for example, has long required "clean-rooms" that
use extensive air filtering to reduce the number and size of
particles in the air to some acceptable level. Other manufacturing
environments have similar but distinct requirements. For example,
in pharmaceutical or medical device manufacturing environments it
is critical to control not only the number of particles in the air,
but minimization of biologic particles is of particular importance.
Microbial contamination, for example, can render an entire batch of
pharmaceutical product unusable leading to significant monetary
losses in the manufacturing process. Additionally, it is
advantageous to have instantaneous detection of contamination
events, including instantaneous information about whether a
contamination event is biologic or non-biologic, during the
manufacturing process for pharmaceuticals or medical devices.
A variety of systems and methods exist that provide instantaneous
detection of fluid borne particles. For example, certain detectors
have been designed to detect fluid borne particles and provide
warning when the number of particles within an air sample exceeds a
predetermined minimum value. Exemplary devices are described in
U.S. Pat. Nos. 5,646,597, 5,969,622, 5,986,555, 6,008,729, and
6,087,947, all to Hamburger et al. These detectors all involve
direction of a light beam through a sample of environmental air
such that part of the beam will be scattered by any particles in
the air, a beam blocking device for transmitting only light
scattered in a predetermined angular range corresponding to the
predetermined allergen size range, and a detector for detecting the
transmittal light. An alarm is actuated if the light detected at
the detector is above a predetermined level.
Additionally, systems and methods exist that instantaneously
determine whether detected particles are biologic or inert. For
example, U.S. Pat. No. 7,430,046 to Jiang et al., discloses systems
and methods for simultaneously measuring particle size by use of
Mie scattering and determining whether the measured particles are
biologic or inert by detecting fluorescence excited in certain
biological chemicals present in the measured particles.
Although fluorescence analysis can, in certain cases, be used to
determine the type of biologic particle detected, i.e., the type of
organism, it would be advantageous to have additional systems and
methods that could simultaneously collect information on particle
size, whether a particle is biologic or non-biologic, and the type
of biologic particle that has been detected.
SUMMARY OF THE INVENTION
Embodiments of the invention continuously sample fluid containing
particles from an environment to be monitored. Sampled fluid is
passed through a sampling area, where it is exposed to light. Light
scattered by the particles in the fluid is detected and used to
determine the size of the particles. Light emitted by fluorescence
from the particles is also detected and used to determine whether
the particles are biological or non-biological. A filter containing
a gas-permeable substance to which microbes adhere, (i.e., a
microbe collection filter) is placed after the sampling area, such
that the microbe collection filter is placed into the flow of the
fluid being measured. In certain embodiments, the filter is
situated atop a circular support plate including perforations
allowing fluid to flow through the plate. The microbe collection
filter collects biological particles from at least a portion of the
flow of fluid. After optical measurements have been performed for a
predetermined amount of time, and microbe collection filter has
been exposed, the filter is removed, microbial nutrients and/or
water are added if necessary, and the filter is incubated and
examined for the growth of organisms according to any of a number
of means, e.g., colony counting, observing the macroscopic
appearance of the growth patterns of microbes on the filter,
microscope observation of the microbes or chemical testing for the
metabolic by-products of microbial growth.
In one embodiment, a microbial detection and identification system
is described. The system includes a sampling area including a
fluid, a light source, a first optical detector and a second
optical detector. The system further includes a microbe collection
filter in fluid communication with the sampling area. The light
source illuminates particles in the sampling area, the first
detector detects light scattered into a predetermined angular range
by particles of a predetermined size, and the second detector
measures light emitted by fluorescence from illuminated biological
particles in the sample area. Additionally, the fluid is exposed to
the microbe collection filter causing particles in the fluid to
adhere to the microbe collection filter.
In certain embodiments, a first optical system directs scattered
light from the sampling area to the first detector and a second
optical system that directs fluorescence light from the sampling
area to the second detector. In certain embodiments, the second
optical system includes a long-pass filter that selectively
transmits light having a wavelength of light emitted by
fluorescence by illuminated particles in the sample area. In
certain embodiments, an ellipsoidal reflector is included having a
first focus at the sampling area and a second focus near the second
detector.
In certain embodiments, the long-pass optical filter comprises two
long-pass reflective filters in series. In certain embodiments, the
light source comprises an LED or a diode laser, and the light
source emits at a wavelength of approximately between 350 nm and
410 nm. In certain embodiments, the microbe collection filter
includes a first surface, a second surface, and a gas permeable
interior allowing the fluid to flow into the first surface, and out
of the second surface. In certain embodiments, the microbe
collection filter is gelatin.
In some embodiments, the fluid is air, and additional embodiments
include a blower in fluid communication with the sampling area,
where the blower supplies negative pressure to the sampling area
thereby drawing environmental air into the sampling area and
evacuating air from the sampling area as it is optically measured.
In certain embodiments, the blower is in fluid communication with
the microbe collection filter, and air is drawn through the microbe
collection filter after it is optically measured.
Certain embodiments recite a method of detecting microbial
contamination in a fluid. The method involves illuminating a fluid
with a light source, detecting particles of a pre-determined size
range present in the fluid by measuring light scattered by
illuminated particles into a predetermined range of angles,
classifying particles in the fluid as biological or non-biological
by measuring fluorescent light emitted from illuminated particles,
and exposing a microbe collection filter to the fluid.
Certain embodiments include storing data related to the detection
of particles of a predetermined size range and classification of
particles as biological or non-biological for further analysis.
Additional embodiments include culturing the exposed microbe
collection filter. Other embodiments include analyzing the cultured
exposed microbe collection filter to determine the types of
microbes present on the microbe collection filter at the time of
exposure.
Certain embodiments include analyzing the cultured exposed microbe
collection filter comprises one or more of: colony counting,
observing the macroscopic appearance of the growth patterns of
microbes on the cultured exposed microbe collection filter,
microscope observation of microbes on cultured exposed microbe
collection filter, and chemical testing for the metabolic
by-products of microbial growth present on the cultured exposed
microbe collection filter. Other embodiments include storing data
related to the detection of particles of a predetermined size range
and classification of particles as biological or non-biological for
further analysis. Certain embodiments include correlating the
determination of the types of microbes present on the microbe
collection filter at the time of exposure with stored data related
to size of particles present in the fluid at the time of exposure
and the status of the particles as biological or
non-biological.
Certain embodiments include a method of characterizing a
contamination event. The method involves illuminating a first fluid
with a light source, detecting particles of a pre-determined size
range present in the first fluid by measuring light scattered by
illuminated particles into a predetermined range of angles, and
classifying particles in the first fluid as biological or
non-biological by measuring fluorescent light emitted from
illuminated particles. The method also includes, storing data
related to a scattering and fluorescence characteristics of
particles in the first fluid, exposing a microbe collection filter
to the first fluid, culturing the microbe collection filter and
analyzing the cultured exposed microbe collection filter to
determine the types of microbes present on the microbe collection
filter at the time of exposure. The method also includes
correlating the determination of the types of microbes present on
the microbe collection filter at the time of exposure with stored
data related to the scattering and fluorescence characteristics of
particles in the first fluid, optically detecting scattering and
fluorescence characteristics of particles in a second fluid, and
comparing the detected scattering and fluorescence characteristics
in the second fluid with the stored data related to the scattering
and fluorescence characteristics of particles in the first fluid.
Other embodiments are directed to a method where on the basis of
the comparison, determining that microbes present in the first
fluid are likely present in the second fluid.
Advantages of the invention include the ability to perform
instantaneous, simultaneous particle sizing and detection of
biological or non-biological organisms. Additional advantages
include the ability to determine the type of biological particle
detected and correlate data on the type of organism detected with
the real-time particle data to fully characterize a contamination
event. Additional advantages include the possibility of predicting
the type of microbial contamination associated with future events
on the basis of past correlations between optical and growth-medium
measurements of particles.
BRIEF DESCRIPTION OF THE DRAWINGS
Implementations of the invention will become more apparent from the
detailed description set forth below when taken in conjunction with
the drawings, in which like elements bear like reference
numerals.
FIG. 1 is a schematic diagram of a system for instantaneous and
simultaneous particle sizing and biological detection according to
an embodiment of the invention.
FIG. 2 is a schematic diagram showing the microbial collector
components of a particle sizing and biological detection system
according to an embodiment of the invention in additional
detail.
FIG. 3 is an exploded drawing showing an assembly for holding a
microbe collection filter according to an embodiment of the
invention.
FIG. 4 is a plan view of an exemplary filter support plate suitable
for use in conjunction with an embodiment of the invention.
FIG. 5 is a schematic flowchart illustrating steps of a method of
microbial detection and analysis according to an embodiment of the
invention.
FIG. 6 is a schematic block diagram of a computer-automated machine
vision and analysis system according to an embodiment of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
This invention is described in preferred embodiments in the
following description with reference to the Figures, in which like
numbers represent the same or similar elements. Reference
throughout this specification to "one embodiment," "an embodiment,"
or similar language means that a particular feature, structure, or
characteristic described in connection with the embodiment is
included in at least one embodiment of the present invention. Thus,
appearances of the phrases "in one embodiment," "in an embodiment,"
and similar language throughout this specification may, but do not
necessarily, all refer to the same embodiment.
The described features, structures, or characteristics of the
invention may be combined in any suitable manner in one or more
embodiments. In the following description, numerous specific
details are recited to provide a thorough understanding of
embodiments of the invention. One skilled in the relevant art will
recognize, however, that the invention may be practiced without one
or more of the specific details, or with other methods, components,
materials, and so forth. In other instances, well-known structures,
materials, or operations are not shown or described in detail to
avoid obscuring aspects of the invention.
The schematic flow charts included are generally set forth as
logical flow chart diagrams. As such, the depicted order and
labeled steps are indicative of one embodiment of the presented
method. Other steps and methods may be conceived that are
equivalent in function, logic, or effect to one or more steps, or
portions thereof, of the illustrated method. Additionally, the
format and symbols employed are provided to explain the logical
steps of the method and are understood not to limit the scope of
the method. Although various arrow types and line types may be
employed in the flow chart diagrams, they are understood not to
limit the scope of the corresponding method. Indeed, some arrows or
other connectors may be used to indicate only the logical flow of
the method. For instance, an arrow may indicate a waiting or
monitoring period of unspecified duration between enumerated steps
of the depicted method. Additionally, the order in which a
particular method occurs may or may not strictly adhere to the
order of the corresponding steps shown.
FIG. 1 shows a system for instantaneous particle sizing and
biological particle detection according to an embodiment of the
invention. The system of FIG. 1 includes a housing 102, which
serves to seal and/or isolate the interior components of the system
from contamination from the surrounding air and interference from
light sources in the environment of the system.
The system of FIG. 1 further includes a light source 105. In one
embodiment, light source 105 produces an output having a wavelength
between 270 nm and 410 nm. In one embodiment, light source 105
produces an output having a wavelength between 350 nm and 410 nm.
In one embodiment, light source 105 produces an output having a
wavelength of about 405 nm. The spectral characteristics of light
source 105 are such that light emitted by light source 105 is
capable of undergoing Mie scattering when interacting with
particles of a size range of interest. Additionally, light source
105 is selected to have a wavelength capable of exciting intrinsic
fluorescence from metabolites inside microbes and other biological
particles. A wavelength of about 270 nm to 410 nm is chosen based
on the observation that microbes and biological particles contain
at least one of a number of primary metabolites that exhibit
fluorescence: tryptophan, which normally fluoresces at excitation
wavelengths of about 270 nm, with a range of about 220 nm to about
300 nm; nicotinamide adenine dinucleoetide (NADH), which normally
fluoresces at excitation wavelengths of about 240 nm, with a range
of about 320 nm to about 420 nm; and riboflavin, which normally
fluoresces at wavelengths of about 200 nm, with a range of between
320 nm to about 420 nm. In the case of bacterial endospores,
dipicolinic acid (DPA) normally fluoresces at excitation
wavelengths of about 400 nm, with a range of about 320 nm to about
420 nm. A light source having a wavelength output of about 350 nm
to about 410 nm ensures excitation of two of the three aforesaid
primary metabolites: NADH and riboflavin, as well as DPA. Selection
of this wavelength band allows for fluorescence to be generated and
detected in bio-particles, but excludes excitation of
non-biological sources of florescence such as diesel engine exhaust
and other inert particles such as dust or baby powder.
Light source 105 can be a laser such as a diode laser, an LED or a
spectrally filtered broadband source such as a lamp. Light source
105 can optionally include collimating or beam shaping optics to
produce a substantially collimated output and/or an output having a
transverse beam profile that is flat, in terms of power.
Optionally, light source 105 includes an optical fiber that
delivers light to the vicinity of the other elements of the system
of FIG. 1. When an optical fiber is used to deliver light from a
remotely situated light source, collimating or beam shaping optics
may optionally be provided at the output of the optical fiber.
Light source 105 provides a substantially collimated beam of light
to at least a portion of the sampling area 110. The intersection of
the substantially collimated beam from light source 110 and the
sampling area creates an interrogation zone, which is an
illuminated portion of the sampling area. In one embodiment, where
the fluid to be measured is air or some other gas, sampling area
110 is defined by the space between two air nozzles that provide
air flow through the sampling area. In the embodiment of FIG. 1,
sampling area 110 is defined by the space between two nozzles, an
entrance nozzle 112, which supplies air to the sampling area, and
an exit nozzle 114, which extracts air from the sampling area.
Upon illumination from light source 105, particles within sampling
area 110 scatter light by Mie scattering. Mie scattering generally
scatters light at angles inversely proportional to particle size.
Accordingly, relatively small particles will scatter light at
higher angles relative to the scattering produced by relatively
larger particles. In practice, scattered light emerges from
sampling area 110 in a cone centered about an axis defined by the
collimated beam emerging from light source 105. The amount of light
scattered into various angles is used, according to certain
embodiments of the invention, to determine the size of the
particles scattering light.
The system of FIG. 1 further includes a scattered light collection
lens 115. In one embodiment, scattered light collection lens is a
plano-convex lens arranged with the plano side facing toward
sampling area 110 to minimize the spherical aberration associated
with collecting and collimating scattered light. Scattered light
collection lens 115 collects and collimates light scattered at
relatively high angles by particles in sampling area 110 by being
configured and positioned such that its front focal plane is
co-incident with sampling area 110.
The system of FIG. 1 further includes a scattered light condenser
lens 120. Scattered light condenser lens 120 takes collimated light
emerging from scattered light collection lens 115 and focuses that
light onto scattered light detector 125, which generates an
electrical signal in proportion to the amount of scattered light
incident on detector 125. In one embodiment, scattered light
detector 125 is a photo-diode.
The system of FIG. 1 further includes beam blocking device 135.
Beam blocking device 135 prevents further propagation of the
collimated beam emitted by light source 105 after light emitted by
light source 105 has propagated through sampling area 110. In one
embodiment, beam blocking device 135 is a disk of optically
absorptive material of a diameter somewhat greater than the beam
diameter of the collimated beam emitted by light source 105. In the
embodiment of FIG. 1, beam blocking device 135 is affixed to
scattered light collection lens 115. In certain embodiments, beam
blocking device 135 is a disc of black anodized aluminum. In other
embodiments, beam blocking device 135 is a small light box with an
absorptive interior coating arranged to force light emitted by
light source 105 and entering the light box to undergo multiple
internal reflections. Alternatively, beam blocking device is a fold
mirror that directs light emitted by light source 105 to a beam
dump arranged at some position outside of the optical components
pictured. Alternatively, beam blocking device 135 is a conical
shape with an absorptive interior coating that the beam enters at
the open side. Beam blocking device 135 can be any device or
combination of devices that prevents further propagation of the
collimated beam, or stray reflections caused by the collimated
beam, emitted by light source 105 after light emitted by light
source 105 has propagated through sampling area 110.
The scattered light detection components of the system of FIG. 1
are arranged such that only a specific range of angles of scattered
light are detected by scattered light detector 125. This can be
accomplished in a number of ways. For example, the scattered light
collection components can be sized and/or positioned in such a way
as to only intercept light that has been scattered into a
predetermined angular range of interest. Beam blocking device 135
inherently blocks very low angle light, including the unscattered
light that passes through the sampling area 110 from light source
105. In one embodiment, beam blocking device 135 is sized to block
not only light that is propagating along an optical axis defined by
the beam from light source 105, but also light that is scattered at
low, but still non-zero angles. In this way, in certain
embodiments, beam blocking device 135 is used to define a lower
bound for the range of angles measured by the scattered light
measurement components.
In certain embodiments, an upper bound for the range of angles
measured by the scattered light measurement components is
established by the position and size of scattered light collection
lens 115. Light scattered at high angles will not be intercepted by
scattered light collection lens 115, so the size and distance of
scattered light collection lens from the plane of sampling area 110
defines an upper bound for the range of angles measured by the
scattered light measurement components. Additionally or
alternatively, annular masks of optically non-transmissive material
may be placed in the scattered light detection path, for example,
on scattered light condenser lens 120 or the scattered light
collection lens 115 to limit the range of scattering angles
measured.
The system of FIG. 1 further includes an ellipsoidal reflector 130.
The shape of ellipsoidal reflector 130 is defined with respect to a
vertex located off the axis defined by the beam of substantially
collimated light emitted by light source 105. In other words, when
viewed with respect to the axis defined by light source 105, the
collimated beam emitted by light source 105, sampling area 110 and
the scattered light collection components, ellipsoidal reflector
130 is an off-axis ellipse. A first focus of ellipsoidal reflector
130 is located at the sampling area 110 and is substantially
co-incident with the front focal point of scattered light collector
lens 115. A second focus of ellipsoidal reflector 130 is located
near the input port of a photomultiplier tube ("PMT") 150, the
function of which is set forth in further detail below. In one
embodiment, ellipsoidal reflector 130 includes a circular aperture,
for example, near its vertex, to allow for uninterrupted
propagation of light from light source 105 to sampling area
110.
Particles undergoing florescent emission within the sampling area
110 will emit light isotropically, that is, will emit equal optical
power into all angles defining a sphere. Ellipsoidal reflector 130
is positioned such that it intercepts at least a portion of the
light emitted by fluorescence from particles within the sampling
area. The fluorescence light collected by parabolic reflector 130
at its first focus is directed along an axis defined by ellipsoidal
reflector 130 toward the second focus located near an input port to
PMT 150.
The system of FIG. 1 further includes back-to-back long-pass
optical filters 140, i.e., two long-pass, reflective interference
filters in series. In one embodiment, long-pass optical filters 140
are reflective interference type filters that transmit light having
a wavelength longer than a certain wavelength while reflecting
light having a wavelength shorter than a certain wavelength. The
spectral characteristics of filters 140 are such that light emitted
by particles within the sampling area 110 by fluorescence is
transmitted, while light having substantially the same wavelength
as that emitted by light source 105 is reflected.
Since fluorescence results in the emission of light having a longer
wavelength than the excitation wavelength, filters 140 pass only
light emitted by fluorescence, while reflecting noise (e.g., stray
reflections) from the light source 105 as well as light scattered
by particles within the sampling area 110 at angles toward PMT 150.
Two long-pass filters are used in series to improve the performance
of the filters. This arrangement is advantageous when placing the
filters 140 in a converging beam, i.e., where the filters are used
at non-zero angles of incidence.
The system of FIG. 1 further includes blower 160. Blower 160 is
arranged to draw fluid in the sampling area out of the system after
it has been optically interrogated.
It is important to note that the specifics of the optical
collection systems for scattered and fluorescent light described
above with respect to FIG. 1 are exemplary and not required. Any
combination or configuration of optical system capable of
simultaneously collecting and measuring scattered and fluorescent
light should be deemed to be within the scope of embodiments of the
invention.
FIG. 2 shows the microbial collection components of a system
according to an embodiment of the invention. In certain
embodiments, the microbial collection components illustrated in
FIG. 2 are used with the optical sizing and biological detection
components described in reference to FIG. 1. FIG. 2 omits the
optical components for clarity, however. The system of FIG. 2
includes a housing 202. In the system of FIG. 2, air to be measured
is routed to a sampling area 210 via an entrance nozzle 212. Once
in sampling area 210, particles in the air are sized and classified
as biological or non-biological by measuring the angular
distribution of scattered light and fluorescence. One exemplary way
this is accomplished by the operation of system components
described above with respect to FIG. 1. After being measured
optically in sampling area 210, air being measured is extracted
from sampling area by exit nozzle 214 where it is passed to filter
holder 260.
Filter holder 260 contains microbe collection filter 265. In one
embodiment, microbe collection filter 265 is a 47 mm gelatin plate
that can be removed from filter holder 260 and placed in a
conventional Petri dish for incubation. Microbe collection filter
265 has a front side facing toward exit nozzle 214 and a rear side
facing toward perforated support plate 266, which itself includes
perforations allowing fluid flowing through microbe collection
filter 265 to flow the perforations into a rear chamber 267 of
filter holder 260. Rear chamber 267 is in fluid communication with
exit fluid line 269. Microbe collection filter 265 is supported by
perforated support plate 266, which includes a plurality of
perforations allowing fluid to flow from the back side of the
microbe collection filter 265 through the support plate 266. Filter
holder 260 is configured to provide an air-tight seal, by the use
of compressed o-rings or the like, such that substantially all of
the air extracted from sampling area 210 through exit nozzle 214
flows through the microbe collection filter 265. From the back side
of microbe collection filter 265, air flows through the
perforations in support plate 266, and through fluid exit line 269,
which is in fluid communication with blower 270. Blower 270 draws
fluid through exit line 269, the perforations in support plate 266,
and microbe collection filter 265. Since microbe collection filter
265 and its support plate provide a partial obstruction to air
flow, the suction created by blower 270 at the front side of
microbe collection filter 265 will be less than the suction created
by blower 270 near the back side of microbe collection filter 265.
The suction at the back side of microbe collection filter 265 is
maintained at a level sufficient to extract the air being measured
from sampling area 210. This is accomplished by selecting
sufficiently high blower suction, and a sufficiently high
cross-sectional perforation area of the gelatin filter's support
plate to maintain adequate negative pressure at the exit nozzle
214.
In one embodiment, microbe collection filter 265 is a gas-permeable
substance that causes microbial particles in fluid exposed to the
filter to adhere to the filter. Additionally, microbe collection
filter 265 is configured to maintain microbial viability so that
microbes collected by the filter can be cultured and analyzed. In
particular embodiments, microbe collection filter 265 is a gelatin
wafer having a sufficiently high water content to maintain
microbial viability, although the use of gelatin is not a
requirement. Any filter containing a gas permeable substance with a
high moisture content to maintain biological viability, to which
microbes present in fluid exposed to the filter adhere, is
acceptable to use as the filter described herein.
In the embodiment of FIG. 2, microbial collection filter 265 is
removable so that, after a measurement period, the microbe
collection filter 265 can be incubated and then analyzed to
determine the number, and in certain embodiments, the types of
microbes that were present in the measured air during the
measurement period. In an exemplary process, after exposure,
microbial collection filter 265 is removed and covered with a
sterile cover for transport. After transport, microbial collection
filter 265 is placed in contact with a growth medium containing
plate, e.g., an agar containing Petri dish. The filter is then
incubated for some amount of time, and the resulting microbial
growth is analyzed. Analysis can take several forms. For example,
microbial colonies may be visually visible and can be counted to
determine the number of microbes that landed on the microbial
collection filter during the measurement period. Additional
analyses may be performed, for example, in certain cases the shape,
i.e., the macroscopic appearance, of a microbial colony can provide
information about the type of microbe present. Additionally or
alternatively, microscopic observation of the microbes, chemical
testing for the metabolic by-products of microbial growth and/or
DNA analysis can determine the type of microbe collected.
FIG. 3 is an exploded drawing showing a microbe collection filter
assembly according to an embodiment of the invention. FIG. 3 shows
a preferred embodiment for the filter holder 260 described above
with respect to FIG. 2. Microbe collection filter assembly includes
cartridge housing 305. Cartridge housing 305 includes hose barb 310
and flow sensor tube 315 which are arranged at sensor input
aperture 320 one the side of the cartridge housing. This sensor
assembly can be used to monitor air flow by the Venturi effect or
the like. Fluid exiting the system enters cartridge housing, and
therefore filter assembly, via input aperture 321 which is in fluid
communication with an exit nozzle of the system, for example, exit
nozzle 214 described above with FIG. 2. After being extracted from
the sampling area of the system, fluid is routed into cartridge
housing 305 via, for example, a non-illustrated hose attached to
hose part 310. Cartridge housing 305 also includes output aperture
322 and hose barb 325. Fluid under test passes through output
aperture 322 to non-illustrated blower after passing through
microbe collection filter, which is described in additional detail
below.
The assembly of FIG. 3 includes cartridge 330, which includes a
knurled handle for easier handling. The function of cartridge 330
is to hold a microbe collection filter in cartridge housing 305
such that the microbe collection filter is sealed in the flow path
of fluid being extracted from the system. Cartridge 330 includes
support plate 335. Support plate 335 is, in one embodiment, a rigid
disk containing perforations that allow fluid flow. An illustrative
embodiment of support plate 335 is set forth in more detail below
with respect to FIG. 4. Support plate 335 is secured to cartridge
330 by a fastener 340 that engages a through-hole in support plate
335.
The assembly of FIG. 3 includes microbe collection filter 345. In
one embodiment, microbe collection filter is a disk of
gas-permeable, water impregnated gelatin, but this is not a
requirement. Microbe collection filter need only be capable of
trapping some microbes present in air to which microbe collection
filter 345 is exposed. Microbe collection filter is secured into
cartridge 330 with o-ring 350 and clamp ring 355. Once assembled
cartridge 330 is inserted into and sealed against cartridge housing
305 with additional o-rings 360, 365. Cartridge 330 is secured in
cartridge housing 305 using a plurality of pins 370. Upon assembly
microbe collection filter 345 is sealed in the fluid flow path
emerging from the system such that all of the fluid emerging from
the system is forced through microbe collection filter 345.
FIG. 4 shows an exemplary filter support plate suitable for use in
conjunction with an embodiment of the invention. In one embodiment,
filter support plate is approximately 47 mm in diameter. The filter
support plate of FIG. 4 includes a central aperture 405 having a
diameter of approximately 3 mm. Central aperture 405 is optionally
chamfered to receive a non-illustrated fastener. Filter support
plate includes an outside annular zone 407, configured to lay
outside the area of microbe collection filter beings supported by
the filter support plate. Outside annular zone 407 is optionally
used for mounting or sealing to the filter support plate. In one
embodiment, the filter support plate of FIG. 4 is approximately 1
mm thick.
The filter support plate of FIG. 4 includes a plurality of
perforations 410 that allow air to flow from front to back.
Although circular, radially arranged perforations are shown with
respect to the filter support plate of FIG. 4, this not a
requirement. The only requirement is that the filter support plate
includes perforations sufficient to allow air to flow through a
gas-permeable microbe collection filter, e.g., a gelatin wafer, in
contact with the filter support plate while mechanically supporting
the microbe collection filter against deformations caused by air
pressure.
FIG. 5 shows a method of detecting and identifying microbes
according to an embodiment of the invention. In the method of FIG.
5 particles in a fluid under test, for example air, are optically
characterized. Optical characterization is conducted by
illuminating the fluid under test with a beam of light, for example
a beam of light generated by a laser or LED emitting at a
wavelength of between 270 nm and 410 nm. Particles present in the
fluid scatter light by Mie scattering at various angles depending
on the size of the scattering particle. Additionally, biological
particles in the fluid absorb light and re-radiate light at longer
wavelengths by fluorescence. Light that is scattered into
predetermined angles by particles in the fluid is detected and used
to determine the size of the scattering particles. At the same
time, light emitted by fluorescence from the particles is detected
and used to make a determination as to whether the particles are
biological or non-biological. The scattered light and fluorescence
measurements are temporally correlated to determine the size of
biological and non-biological particles present in the fluid being
measured. Additionally, the scattered light and fluorescence
measurements are stored, for example as a function of time, for
later use and analysis.
After the fluid being measured is optically characterized, that
fluid is exposed to a gas-permeable microbe collection filter,
e.g., a gelatin wafer. After a predetermined period of time, the
gas-permeable microbe collection filter is removed and cultured to
encourage microbial growth. The culturing process comprises
providing nutrients to microbes on the exposed gas-permeable
microbe collection filter, and incubating same. After an incubation
period has elapsed, the cultured microbe collection filter is
analyzed. Such analysis can occur by any number of means, for
example, colony counting, observing the macroscopic appearance of
the growth patterns of microbes on the filter, microscope
observation of the microbes, chemical testing for the metabolic
by-products of microbial growth, or DNA testing.
If the analysis applied to the incubated filter determines the
number of microbes that were present in the filter after the
exposure period, this number is, in one embodiment, correlated with
the optically measured data on the size and number of biological
particles detected during the exposure period. Accordingly,
correlating the optically measured data with the data from analysis
of the cultured filter can be as simple as comparing the microbe
count from the filter with the number of optically detected
particles.
If the analysis applied to the incubated filter determines the
types of microbes present on the filter at the time of exposure,
this information, in one embodiment, is correlated with the stored
data on the size and biological status of particles measured
optically at the time the filter was exposed. This correlation
optionally results in a determination of the number and types of
microbes present in the fluid sample during the measurement period,
i.e., the period of time the filter was exposed to fluid for which
optical data was collected. This determination is made with the aid
of conventional knowledge of the size of the types microbes
identified on the filter by the analysis of the incubated
filter.
The method illustrated in FIG. 5 has a number of advantages. First,
the method of FIG. 5 provides a more complete retrospective
characterization of the measured fluid sample by including
information about the type of microbes that were instantaneously
detected. Second, data collected according to the method of FIG. 5
allows for tentative, real-time characterization of future
biological contamination events based on previously measured data.
For example, suppose a contamination event occurs that results in
numerous biological particles in the 0.4-0.8 .mu.m range, which are
instantaneously detected by the optical systems and methods set
forth above. Upon analysis of a growth-medium containing filter
exposed to air measured during this hypothetical contamination
event, it is determined that most of these biological particles
were a particular kind of microbe. In the future, whenever a spike
of biological particles in the 0.4-0.8 .mu.m range is detected
under similar conditions, it can be assumed that another
contamination event associated with the previously detected microbe
is occurring in the monitored environment, and a real-time response
appropriate to that contamination event can be coordinated. A
predictive step is illustrated in FIG. 5, where microbial
characteristics of a contamination event are predicted by matching
optically collected data with previously optically collected data
that was previously correlated to a particular set of identified
microbes.
Computer hardware, software and machine vision components can be
used to perform, assist or simplify and of the process steps
performed herein. FIG. 6 shows an exemplary computer system for
performing analysis of a fluid under test according to an
embodiment of the invention. The system of FIG. 6 includes a
particle sizing, biological particle identification, and microbial
collection system 605. In certain embodiments, system 605 is the
system described above with respect to FIGS. 1 and 2. System 605
performs real-time particle detection and sizing as well as
real-time determination of whether detected particles are
biological or non-biological. Additionally, system 605 performs
microbial collection by exposure of a microbe collection filter to
the fluid being optically measured. The optically measured data is
converted to electrical signals by electronics included with system
605, for example, by the drive electronics associated with a
photo-diode measuring optical scattering and a PMT measuring
fluorescence light. The electrical signals generated by these two
detectors are transmitted from system 605 to optional computer data
acquisition card 610, which is electronic communication with
computer 615. Optically measured data is then stored in a
persistent storage medium, for example, hard disk 620.
The system of FIG. 6 further includes machine vision camera 630,
which is electronic communication with image capture board 625,
which in turn is in electronic communication with computer 615.
After a microbe collection filter 635 has been incubated, and
microbial colonies are visible, camera 630 captures an image of the
colonies visible on filter 635. To assist in this task, optional
non-illustrated steppers may be used to translate the field of view
of camera 630 with respect to filter 635 or vice-versa. An image or
images of the filter showing microbial colonies is stored by
computer 615 to disk 620.
Disk 620 also includes computer readable instructions 622 operable
to cause computer 615 to correlate and/or compare the colony count
detected by camera 630 with the optically measured data regarding
the number and biological or non-biological status of particles
measured during the time period when filter 635 was exposed. More
generally, embodiments of the invention include instructions, such
as instructions 622, residing in computer readable medium, such as
for example computer hard drive 620 wherein those instructions are
executed by a processor, such as processor residing in computer
615, to perform one or more of steps illustrated with respect to
FIG. 5, for example the storage step, analyze step, correlate step
or predict step. Computer readable instructions 622 need not reside
on hard disk 620, but may reside in any medium capable of being in
electronic communication with a processor capable of executing
instructions 622. For example, there is no requirement that data
(e.g., optically measured particle data) be stored on the same
medium that includes instructions 622. Additionally, while FIG. 6
shows computer 620, camera 630 and other components being separate
from system 605, this is not a requirement. System 605 could be
configured to include a microprocessor, storage, memory,
input/output electronics and a camera necessary to perform the
method steps described herein.
In other embodiments, Applicants' invention includes instructions
residing in any other computer program product, where those
instructions are executed by a computer external to, or internal
to, systems such as system 605, to perform one or more steps
described with respect to FIG. 5. In either case, the instructions
may be encoded in computer readable medium comprising, for example,
a magnetic information storage medium, an optical information
storage medium, an electronic information storage medium, and the
like. By "electronic storage media," Applicants mean, for example
and without limitation, one or more devices, such as and without
limitation, a PROM, EPROM, EEPROM, Flash PROM, compactflash,
smartmedia, and the like.
While the preferred embodiments of the present invention have been
illustrated in detail, it should be apparent that modifications and
adaptations to those embodiments may occur to one skilled in the
art without departing from the scope of the present invention as
set forth in the following claims.
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